RNA-dependent RNA polymerase encoded by hepatitis C virus: biomedical applications

The hepatitis C viruses (HCVs) are a group of small enveloped RNA viruses that have been viewed as a leading cause of chronic hepatitis in humans. Infections by HCV represent a serious global health problem, because millions of people worldwide are infected and no efficient treatment is available at the present time. Since HCV was identified in 1989, considerable effort has been devoted to the discovery and development of novel molecules to treat HCV-related diseases. One of the approaches is the development of novel inhibitors that interrupt the normal functions of HCV NS5B, an RNA-dependent RNA polymerase essential to HCV replication. This review summarizes recent advances in the biochemical and structural understanding of HCV NS5B polymerase as well as in the development of antiviral agents targeting this important enzyme. Received 19 March 2002; received after revision 23 April 2002; accepted 23 April 2002     

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5′-hydroxyl group and a 2′,3′-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed. Received 21 June 2001; received after revision 18 July 2001; accepted 20 July 2001     

Crystallization of RNA

Even as the number of RNA structures determined and under study multiplies, the critical step in X-ray diffraction analysis, growth of single well-ordered crystals, remains at the boundary between art and science. Recent advances in methods of RNA synthesis, purification, and characterization, as well as empirical and technical improvements in crystallization techniques, the development of cryo-crystallography, and the wider availability of bright, tunable, X-rays from synchrotron sources are improving the chances of obtaining RNA crystals suitable for X-ray structural analysis. In this review, we summarize the current status of the design, preparation, purification, and analysis of RNA for crystallization and describe the latest approaches to obtaining diffraction-quality crystals. Received 17 October 2000; revised 6 December 2000; accepted 8 December 2000     

Tumor necrosis factor as an antineoplastic agent: pitfalls and promises

The discovery and cloning of the cytokine tumor necrosis factor α (TNF) gave rise to new hopes for a significant victory in the war against cancer. Preclinical in vitro studies in cell cultures and in vivo studies in animal models demonstrated the antitumor capacities of TNF. Although clinical studies were largely made possible by the availability of recombinant TNF, phase I and II clinical trials showed very quickly that the systemic administration of TNF induced severe side effects mainly due to its pleiotropic action on immunocompetent cells. The clinical manifestations of the side effects were similar to those observed during a severe infection and inflammation. Very recently, lessons from these clinical studies yielded refined approaches whereby the toxicity of TNF is limited through local administration, a combination with other therapeutic regimens and targeted gene therapy. These new approaches are slated for larger clinical trials and in the near future might demonstrate the limited but powerful usefulness of TNF as an antineoplastic agent for different types of cancer. Received 7 September 1998; received after revision 15 October 1998; accepted 15 October 1998     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins. Received 30 September 2005; received after revision 5 November 2005; accepted 28 November 2005     

Protecting the terminus: t-loops and telomere end-binding proteins

Telomeric DNA is composed of a region of duplex telomeric tract followed by a single-strand overhang on the 3 G-rich strand. The DNA is packaged by proteins that associate directly with the single- and double-strand regions of the telomeric tract and by their associated proteins. This review discusses the evidence that G-strand overhangs are present on both ends of eukaryotic chromosomes and the steps needed to generate these overhangs. The overhangs are protected by specialized G-overhang-binding protein and/or invasion by the overhang of the duplex region of the telomeric tract to form a structure called a t-loop. The G-overhang-binding proteins identified from different species are described, and their properties compared. The data supporting the existence of t-loops at native telomeres is discussed, and the conditions required to promote their in vitro formation are presented.     

Deoxyribozymes: new activities and new applications

DNA in its single-stranded form has the ability to fold into complex three-dimensional structures that serve as highly specific receptors or catalysts. Only protein enzymes and ribozymes are known to be responsible for biological catalysis, but deoxyribozymes with kinetic parameters that rival ribozymes can be created in the laboratory. Some of these engineered DNA catalysts are showing surprising potential as therapeutic agents, which makes them biologically relevant if not biologically derived. If DNA's natural role is strictly genomic, how significant is its innate catalytic prowess? New examples of engineered deoxyribozymes serve as empirical examples of the potential for catalysis by DNA. These results indicate that the true catalytic power of DNA is limited by discovery and not by chemistry.     

Structure and function of eukaryotic NAD(P)H:nitrate reductase

Pyridine nucleotide-dependent nitrate reductases (NRs; EC 1.6.6.1–3) are molybdenum-containing enzymes found in eukaryotic organisms which assimilate nitrate. NR is a homodimer with an ∼100 kDa polypeptide which folds into stable domains housing each of the enzyme's redox cofactors—FAD, heme-Fe molybdopterin (Mo-MPT) and the electron donor NAD(P)H—and there is also a domain for the dimer interface. NR has two active sites: the nitrate-reducing Mo-containing active site and the pyridine nucleotide active site formed between the FAD and NAD(P)H domains. The major barriers to defining the mechanism of catalysis for NR are obtaining the detailed three-dimensional structures for oxidized and reduced enzyme and more in-depth analysis of electron transfer rates in holo-NR. Recombinant expression of holo-NR and its fragments, including site-directed mutagenesis of key acative site and domain interface residues, are expected to make large contributions to this effort to understand the catalytic mechanism of NR.     

APC/C regulation of axonal growth and synaptic functions in postmitotic neurons: the Liprin-{\varvec \alpha} connection

Protein ubiquitination has critical roles in neuronal physiology and defects in protein ubiquitination have been implicated in neurodegenerative pathology. The anaphase-promoting complex/cyclosome (APC/C) is one of two key E3 ubiquitin ligase complexes that functions in regulating cell cycle transitions in proliferating cells by acting on cyclins and components of the mitotic/meiotic apparatus. Documentation of APC/C's action beyond cell division is sparse. In the past year, however, novel and surprising roles for APC/C in postmitotic neurons, particularly in the modulation of axonal growth and synaptic functions, have been revealed. APC/C and its activator Cdh-1 are found in good abundance in neurons, and these seem to function at different cellular locations, modulating apparently diverse processes such as axonal growth and synaptic function. Interestingly, there also appears to be a single link to these apparently divergent actions of APC/C in neurons--the multi-domain, multi-functional scaffolding protein Liprin-alpha which is an APC/C substrate.